CellularELISAProtocol

FormalinFixedCellPlates1.Trypsinizeconfluentflasks2.Poolandcountcells3.Centrifugeat1500rpmfor10minutes4.ResUSPendtotheappropriateconcentrationincompletemedium4x105cells/mlforepithelialcells2x105cells/mlforfibroblastcells5.Add100μl/cellto96wellcultureplates.6.Incubateovernightat37oC.7.WashplatestwicewithPBS8.Add125μl/well10%BufferedFormalin9.Fixfor15minutesatroomtemperature10.Washthreetimeswithdi-H2O.11.Blotdry.12.Storeat2-8oC.Reagents1.PBS:1%BSA2.PBS:2%BSA3.CarbonateBuffer1.59gNa2CO32.93gNaHCO3Dissolvein900mldi-H2O.CheckpHandadjustto9.6necessary.Qs.to1liter.4.10XSubstrateBuffer,pH6.036.6gCitricAcid,monohydrate113.5gPotassiumdibasicphosphateDissolvein900mldi-H2O.CheckpHandadjustto6.0ifnecessary.Qs.to1liter.5.0.3%H2O2Dilute30%stockPeroxide1:100indi-H2O.6.OPDStock,4.0%4gOPDin100mldi-H2O.Aliquotandstoreat-20oC.Protectfromlight.4.5NH2SO412.0mlConcentratedSulfuricAcid88.0mldi-H2O

Procedure1.WashELISAplatesoncewithdi-H2O.2.Add250μl/wellPBS:2%BSA.3.Incubate1hourat37oC.4.Wash3timeswithdi-H2O.5.Add50μl/wellsupe,ascites,orcontrolsdilutedinPBS:1%BSA.6.Incubatefor2hrat37oC.7.Wash5timeswithdi-H2O.8.Add50μl/wellanti-mouseIgG:HRPdilutedinPBS:1%BSA.9.Incubatefor1hrat37oC.10.Wash5timeswithdi-H2O.Washoncewithcarbonatebuffer.11.Add50μl/wellworkingsubstratesolution0.5ml4.0%OPD5μl30%H2O21.0ml10XSubstratebuffer8.5mldi-H2O.12.Incubatefor20minutesatroomtemperature.13.Add25μl/well4.5NSulfuricAcid14.ReadA490Notes1.Testallsupernatantsat1:5dilution.Testascitesat1:100

ELISA相关试剂设备

• 洗板机
• 化学发光检测仪
• 酶标仪
• ELISA检测试剂盒
• ELISPOT检测
• ELISA试剂盒
• 抗体
• 酶标板

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